RNA interference (RNAi) is an evolutionarily conserved, sequence specific mechanism triggered by double stranded RNA (dsRNA) that induces degradation of complementary target single stranded mRNA and “silencing” of the corresponding translated sequences (McManus and Sharp, Nature Rev. Genet. 3:737 (2002)). RNAi functions by enzymatic cleavage of longer dsRNA strands into biologically active “short-interfering RNA” (siRNA) sequences of about 21-23 nucleotides in length (Elbashir, et al., Genes Dev. 15:188 (2001)). siRNA can be used downregulate or silence the translation of a gene product of interest. For example, it is desirable to downregulate genes associated with various diseases and disorders.
Delivery of siRNA remains problematic (see, e.g., Novina and Sharp, Nature 430::161-163 (2004); and Garber, J. Natl. Cancer Inst. 95(7):500-2 (2003)). An effective and safe nucleic acid delivery system is required for siRNA to be therapeutically useful. Naked dsRNA administered to most subjects will: (1) be degraded by endogenous nucleases; and (2) will not be able to cross cell membranes to contact and silence their target gene sequences.
Viral vectors are relatively efficient gene delivery systems, but suffer from a variety of safety concerns, such as potential for undesired immune responses. Furthermore, viral systems are rapidly cleared from the circulation, limiting transfection to “first-pass” organs such as the lungs, liver, and spleen. In addition, these systems induce immune responses that compromise delivery with subsequent injections. As a result, nonviral gene delivery systems are receiving increasing attention (Worgall, et al., Human Gene Therapy 8:37 (1997); Peeters, et al., Human Gene Therapy 7:1693 (1996); Yei, et al., Gene Therapy 1: 192 (1994); Hope, et al., Molecular Membrane Biology 15:1 (1998)).
Plasmid DNA-cationic liposome complexes are currently the most commonly employed nonviral gene delivery vehicles (Felgner, Scientific American 276:102 (1997); Chonn, et al., Current Opinion in Biotechnology 6:698 (1995)). For instance, cationic liposome complexes made of an amphipathic compound, a neutral lipid, and a detergent for transfecting insect cells are disclosed in U.S. Pat. No. 6,458,382. Cationic liposome complexes are also disclosed in U.S. Patent Application Publication No. 2003/0073640. Cationic liposome complexes, however, are large, poorly defined systems that are not suited for systemic applications and can elicit considerable toxic side effects (Harrison, et al., Biotechniques 19:816 (1995); Li, et al., The Gene 4:891 (1997); Tam, et al, Gene Ther. 7:1867 (2000)). As large, positively charged aggregates, lipoplexes are rapidly cleared when administered in vivo, with highest expression levels observed in first-pass organs, particularly the lungs (Huang, et al., Nature Biotechnology 15:620 (1997); Templeton, et al., Nature Biotechnology 15:647 (1997); Hofland, et al., Pharmaceutical Research 14:742 (1997)).
Other liposomal delivery systems include, for example, the use of reverse micelles, anionic and polymer liposomes as disclosed in, e.g., U.S. Pat. No. 6,429,200; U.S. Patent Application No. 2003/0026831; and U.S. Patent Application Nos. 2002/0081736 and 2003/0082103, respectively.
Recent work has shown that nucleic acids can be encapsulated in small (about 70 nm diameter) “stabilized plasmid-lipid particles” (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle (Wheeler, et al., Gene Therapy 6:271 (1999)). These SPLPs typically contain the “fusogenic” lipid dioleoylphosphatidylethanolamine (DOPE), low levels of cationic lipid (i.e., 10% or less), and are stabilized in aqueous media by the presence of a poly(ethylene glycol) (PEG) coating. SPLP have systemic application as they exhibit extended circulation lifetimes following intravenous (i.v.) injection, accumulate preferentially at distal tumor sites due to the enhanced vascular permeability in such regions, and can mediate transgene expression at these tumor sites. The levels of transgene expression observed at the tumor site following i.v. injection of SPLP containing the luciferase marker gene are superior to the levels that can be achieved employing plasmid DNA-cationic liposome complexes (lipoplexes) or naked DNA.
However, there remains a strong need in the art for novel and more efficient methods and compositions for introducing nucleic acids, such as siRNA, into cells. The present invention addresses this and other needs.